Immunoconjugates and humanized antibodies specific for B-cell lymphoma and leukemia cells

ABSTRACT

A chimeric LL2 monoclonal antibody is described in which the complementarity determining regions (CDRs) of the light and heavy chains of the murine LL2 anti-B-lymphoma, anti-leukemia cell monoclonal antibody has been recombinantly joined to the human kappa and IgG 1  constant region domains, respectively, which retains the immunospecificity and B-cell lymphoma and leukemia cell internalization capacity of the parental murine LL2 monoclonal antibody, and which has the potential of exhibiting reduced human anti-mouse antibody production activity. A humanized LL2 monoclonal antibody is described in which the CDRs of the light and heavy chains have been recombinantly joined to a framework sequence of human light and heavy chains variable regions, respectively, and subsequently linked to human kappa and IgG 1  constant region domains, respectively, which retains the immunospecificity and B-lymphoma and leukemia cell internalization capacities of the parental murine and chimeric LL2 monoclonal antibodies, and which has the potential for exhibiting reduced human anti-mouse antibody production activity. Vectors for producing recombinant chimeric and humanized chimeric monoclonal antibodies are provided. Isolated DNAs encoding the amino acid sequences of the LL2 variable light and heavy chain and CDR framework regions are described. Conjugates of chimeric and humanized chimeric LL2 antibodies with cytotoxic agents or labels find use in therapy and diagnosis of B-cell lymphomas and leukemias.

CROSS-REFERENCE TO RELATED PATENT APPLICATIONS

[0001] This application is a continuation of U.S. Ser. No. 09/741,843filed on Dec. 22, 2000, which is a continuation of U.S. Ser. No.09/127,902 filed on Aug. 3, 1998, now U.S. Pat. No. 6,187,287, which isa continuation of U.S. Ser. No. 08/690,102 filed on Jul. 31, 1996, nowU.S. Pat. No. 5,789,554, which is a continuation of U.S. Ser. No.08/289,576 filed on Aug. 12, 1994, now abandoned. This applicationclaims only subject matter disclosed in the parent applications andtherefore presents no new matter.

BACKGROUND OF THE INVENTION

[0002] The invention relates generally to immunoconjugates fordiagnostic and therapeutic uses in cancer. In particular, the inventionrelates to recombinantly produced chimeric and humanized monoclonalantibodies directed against B-cell lymphoma and leukemia cells, whichantibodies can be covalently conjugated to a diagnostic or therapeuticreagent without loss of antibody binding and internalization functionand with reduced production of human anti-mouse antibodies.

[0003] Non-Hodgkins lymphoma (NHL) and chronic lymphocytic leukemia areB-cell malignancies that remain important contributors to cancermortality. The response of these malignancies to various forms oftreatment is mixed. They respond reasonably well to chemotherapy, and,in cases where adequate clinical staging of NHL is possible, as forpatients with localized disease, satisfactory treatment may be providedusing field radiation therapy (Hall et al., Radiology for theRadiologist, Lippincott, Philadelphia, 1989, pp 365-376). However, thetoxic side effects associated with chemotherapy and the toxicity to thehematopoietic system from local, as well as whole body, radiotherapy,limits the use of these therapeutic methods. About one-half of thepatients die from the disease (Posner et al., Blood, 61: 705 (1983)).

[0004] The use of targeting monoclonal antibodies conjugated toradionuclides or other cytotoxic agents offers the possibility ofdelivering such agents directly to the tumor site, thereby limiting theexposure of normal tissues to toxic agents (Goldenberg, Semin. Nucl.Med., 19: 332 (1989)). In recent years, the potential of antibody-basedtherapy and its accuracy in the localization of tumor-associatedantigens have been demonstrated both in the laboratory and clinicalstudies (see., e.g., Thorpe, TIBTECH, 11: 42 (1993); Goldenberg,Scientific American, Science & Medicine, 1: 64 (1994); Baldwin et al.,U.S. Pat. Nos. 4,925,922 and 4,916,213; Young, U.S. Pat. No. 4,918,163;U.S. Pat. No. 5,204,095; Irie et al., U.S. Pat. No. 5,196,337; Hellstromet al., U.S. Pat. Nos. 5,134,075 and 5,171,665). In general, the use ofradiolabeled antibodies or antibody fragments against tumor-associatedmarkers for localization of tumors has been more successful than fortherapy, in part because antibody uptake by the tumor is generally low,ranging from only 0.01% to 0.001% of the total dose injected (Vaughan etal., Brit. J. Radiol., 60: 567 (1987)). Increasing the concentration ofthe radiolabel to increase the dosage to the tumor is counterproductivegenerally as this also increases exposure of healthy tissue toradioactivity.

[0005] LL-2 (EPB2) is a highly specific anti-B-cell lymphoma andanti-lymphocytic leukemia cell murine monoclonal antibody (mAb) that israpidly internalized by such cells and that can overcome some of theaforementioned difficulties (Shih et al., Int. J. Cancer, 56: 538(1994)). LL2, which is of the IgG2a antibody type, was developed usingthe Raji B-lymphoma cell line as the source of antigen(Pawlak-Byczkowska et al., Cancer Res., 49: 4568 (1989)). Murine LL2(mLL2) is known to react with an epitope of CD22 (Belisle et al., ProcAmer. Assn. Clin. Res., 34: A2873 (1993)). CD22 molecules are expressedin the cytoplasm of progenitor and early pre-B cells, and appear in thecell surface of mature B-cells.

[0006] By immunostaining of tissue sections, mLL2 was shown to reactwith 50 of 51 B-cell lymphomas tested. mLL2 is a highly sensitive meansof detecting B-cell lymphoma cell in vivo, as determined by aradioimmunodetection method (Murthy et al., Eur. J. Nucl. Med., 19: 394(1992)). The Fab′ fragment of mLL2 labeled with ^(99m)Tc localized to 63of 65 known lesions in Phase II trial patients with B-cell lymphoma(Mills et al., Proc. Amer. Assn. Cancer Res., 14: A2857 (1993)). Inaddition, ¹³¹I-labeled mLL2 was therapeutically effective in B-celllymphoma patients (Goldenberg et al., J. Clin. Oncol., 9: 548 (1991)).mLL2 Fab′ conjugated to the exotoxin PE38KDEL induced completeremissions of measurable human lymphoma xenografts (CA-46) growing innude mice (Kreitman et al., Cancer Res., 53: 819 (1993)).

[0007] The clinical use of mLL2, just as with most other promisingmurine antibodies, has been limited by the development in humans of aHAMA response. While a HAMA response is not invariably observedfollowing injection of mLL2, in a significant number of cases patientsdeveloped HAMA following a single treatment with mLL2. This can limitthe diagnostic and therapeutic usefulness of such antibody conjugates,not only because of the potential anaphylactic problem, but also as amajor portion of the circulating conjugate may be complexed to andsequestered by the circulating anti-mouse antibodies. This isexemplified by one study in which about 30% of the patients developedlow level HAMA response following a single injection of about 6 mg ofmLL2 ¹³¹I-IgG and nearly all developed a strong HAMA response withadditional injections. On the other hand, with mLL2 Fab′ labeled with^(99m)Tc, no HAMA response was observed. Such HAMA responses in generalpose a potential obstacle to realizing the full diagnostic andtherapeutic potential of the mLL2 antibody.

[0008] Although, as noted above, the use of fragments of mLL2, such asF(ab′)2 and Fab′, partially alleviate/circumvent these problems ofimmunogenicity, there are circumstances in which whole IgG is moredesirable, such as when induction of cellular immunity is intended fortherapy, or where an antibody with enhanced survival time is required.

[0009] In order to maximize the value of the mLL2 IgG antibody as atherapeutic or diagnostic modality and increase its utility in multipleand continuous administration modalities, it is an object of thisinvention to produce a mouse/human chimeric mAb (cLL2) and a humanizedmAb (hLL2) related to mLL2 that retain the antigen-binding specificityof mLL2, but that elicit reduced HAMA in a subject receiving same.

[0010] It is another object of this invention to provide DNA sequencesencoding the amino acid sequences of the variable regions of the lightand heavy chains of the cLL2 and hLL2 mAbs, including thecomplementarity determining regions (CDR).

[0011] It is also an object of this invention provide conjugates of thehLL2 and cLL2 mAbs containing therapeutic or diagnostic modalities.

[0012] It is a further object of this invention to provide methods oftherapy and diagnosis that utilize the humanized and chimeric mAbs ofthe invention.

[0013] These objects have been achieved by the invention described belowin the specification and appended claims.

SUMMARY OF THE INVENTION

[0014] In one aspect of the invention, there is provided a cLL2 mAbrelated to mLL2 mAb, in which the murine light (VK) and heavy (VH) chainvariable regions are joined to the human constant light (kappa) andheavy (IgG₁) chains.

[0015] This chimeric mAb retains the B-lymphoma and leukemia celltargeting and internalization properties of the parental mLL2.

[0016] In another aspect of the invention, there is provided a hLL2 mAbrelated to mLL2 mAb, in which the complementarity-determining regions(CDRs) of the light and heavy chains of the mLL2 mAb are joined to theframework (FR) sequence of human VK and VH regions, respectively, andsubsequently to the human kappa and IgG₁ constant region domains,respectively. This humanized antibody retains the B-lymphoma andleukemia cell targeting and internalization characteristics of theparental mLL2 mAb, and can exhibit a lowered HAMA reaction.

[0017] In still another aspect, there is provided isolatedpolynucleotides comprising DNA sequences encoding the amino acidsequences of the variable light and heavy chains, respectively, of thehLL2 and cLL2 mAbs.

[0018] In an additional aspect, there is provided the amino acidsequences of the CDRs of the VK and VH chains.

[0019] In yet another aspect, there are provided conjugates in which thehLL2 or cLL2 mAb is covalently bonded to a diagnostic or therapeuticreagent.

[0020] In still another aspect, there are provided methods whereby theaforementioned mAb conjugates can be used to diagnose or treat B-celllymphomas and lymphocytic leukemias.

[0021] These and other aspects and embodiments of the invention willbecome apparent by reference to the following specification and appendedclaims.

DESCRIPTION OF THE FIGURES

[0022]FIG. 1 is a comparison of the murine with the humanized LL2 VK(FIG. 1A) and VH (FIG. 1B) domains. Only hFR sequences (designated asREIHuVK and EUHuVH) different than mFR sequences (designated as murine)are shown, and designated by asterisks. More residues in these positionswere retained in the humanized structure. CDRs are boxed. FR residuesshowing CDR contacts by computer modeling are underlined.

[0023]FIG. 2 shows vicinal relationships of the LL2 CDRs to theirframework regions (FRs). Separate energy-minimized models for the VL andVH domains of mLL2 were constructed, and all FR residues within a radiusof 4.5 Å or any CDR atom were identified as potential CDR-FR contacts.CDRs of the light (L1, L2, and L3, FIG. 2A) and heavy (H1, H2, and H3,FIG. 2B)) chains are shown as “ball and stick” representationssuperimposed on their respective, space-filling FRs.

[0024]FIG. 3 shows the light chain (FIG. 3A) staging (VKpBR) andmammalian expression (pKH) vectors, and the heavy chain (FIG. 3B)staging (VHpBS) and mammalian expression (pG1g) vectors.

[0025]FIG. 4 shows the double-stranded DNA and amino acid sequences ofthe LL2 VK domain (FIG. 4A) and the LL2 VH domain (FIG. 4B). Amino acidsequences encoded by the corresponding DNA sequences are given as oneletter codes. CDR amino acid sequences are boxed. The Asn-glycosylationsite located in FR1 of LL2VK (FIG. 4A) is shown as the underlined NVTsequence.

[0026]FIG. 5A shows the double stranded DNA and corresponding amino acidresidues of the hLL2 VK domain. CDR amino acid sequences are boxed. Thecorresponding data for the VH domain is shown in FIG. 5B.

[0027]FIG. 6 is a schematic diagram representation of the PCR/genesynthesis of the humanized VH region and the subcloning into the stagingvector, VHpBS.

[0028]FIG. 7 shows SDS-PAGE analysis of mLL2 and cLL2 antibodies undernon-reducing (lanes 6-8) and reducing (lanes 3-5, light and heavychains) conditions. Lanes 3 and 6 include a control antibody.

[0029]FIG. 8 shows SDS-PAGE analysis of different versions of cLL2 andhLL2 antibodies under reducing (lanes 3-5) and non-reducing (lanes 6-8)conditions.

[0030]FIG. 9 shows SDS-PAGE analysis on mix-and-match cLL2 and hLL2antibodies under reducing (lanes 3-6) and non-reducing (lanes 7-10)conditions. cLL2 serves as the control.

[0031]FIG. 10 shows the results of a comparative Raji cell competitiveantibody binding assay involving mLL2 and cLL2 antibodies competing forbinding to cells against tracer radiolabeled mLL2.

[0032]FIG. 11 shows the results of a comparative Raji cell competitiveantibody binding assay in which mixed humanized/chimeric LL2s werecompared to cLL2 (FIG. 11A), and two versions of hLL2 compared to cLL2(FIG. 11B).

[0033]FIG. 12 shows a comparison of antibody internalization:surfacebinding ratios as a function of time for cLL2, cLL2 (Q to Vmutagenesis), hLL2 and mLL2 antibodies.

[0034]FIG. 13 shows an SDS-PAGE analysis of mLL2 and cLL2 afterdeglycosylation by endoglycosidase F.

[0035]FIG. 14 shows the effect of deglycosylation of mLL2 on its bindingaffinity to Raji cells.

DETAILED DESCRIPTION OF THE INVENTION

[0036] cDNAs encoding the VL and VH regions of the mLL2 mAb have beenisolated and separately recombinantly subcloned into mammalianexpression vectors containing the genes encoding kappa and IgG₁ constantregions, respectively, of human antibodies. Cotransfection of mammaliancells with these two recombinant DNAs expressed a cLL2 mAb that, likethe parent mLL2 mAb, bound avidly to, and was rapidly internalized by,B-lymphoma cells.

[0037] The CDRs of the VK and VH DNAs have been similarly recombinantlylinked to the framework (FR) sequences of the human VK and VH regions,respectively, which are subsequently linked, respectively, to the humankappa and IgG₁ constant regions, so as to express in mammalian cells asdescribed above hLL2.

[0038] In this specification, the expressions “cLL2” or “cLL2 mAb” areintended to refer to the chimeric monoclonal antibody constructed byjoining or subcloning the murine VK and VH regions to the human constantlight and heavy chains, respectively. The expressions “hLL2” or “hLL2mAb” are intended to refer to the humanization of the chimericmonoclonal antibody by replacing the murine FR sequences in cLL2 withthat of human framework regions.

[0039] Covalent conjugates between cLL2 and hLL2 mAbs and a diagnosticor chemotherapeutic reagent, formulated in pharmaceutically acceptablevehicles (see, e.g., Remington's Pharmaceutical Sciences, 18th ed., MackPublishing Co., Easton, Pa., 1990) can be prepared that have theadvantages, compared to prior art antibody conjugates, of B-celllymphoma-specific and leukemia cell-specific targeting, rapidinternalization into target cells, rapid liberation of the diagnostic orchemotherapeutic reagent intracellularly (thereby increasingeffectiveness of the reagent), and a potential reduction of the HAMAresponse in the human patient.

[0040] As the VK-appended carbohydrate moiety of the cLL2 mAb is shownherein not to be involved in binding to B-lymphoma cells, it ispreferred to use conjugates in which the reagent is bound to theantibody through such carbohydrate moieties, such as through oxidizedcarbohydrate derivatives. Methods for the production of such conjugatesand their use in diagnostics and therapeutics are provided, for example.in Shih et al., U.S. Pat. No. 5,057,313, Shih et al., Int. J. Cancer 41:832 (1988), and copending, commonly owned Hansen el al., U.S. Ser. No.08/162,912, the contents of which are incorporated herein by reference.Direct linkage of the reagent to oxidized carbohydrate without the useof a polymeric carrier is described in McKearn et al., U.S. Pat. No.5,156,840, which is also incorporated by reference.

[0041] A wide variety of diagnostic and therapeutic reagents can beadvantageously conjugated to the antibodies of the invention. Theseinclude: chemotherapeutic drugs such as doxorubicin, methotrexate,taxol, and the like; chelators, such as DTPA, to which detectable labelssuch as fluorescent molecules or cytotoxic agents such as heavy metalsor radionuclides can be complexed; and toxins such as Pseudomonasexotoxin, and the like. Several embodiments of these conjugates aredescribed in the examples below.

[0042] Cell lines and culture media used in the present inventioninclude LL2 (EPB-2) hybridoma cells (Pawlak-Byczkowska et al. 1989above), Sp2/0-Ag14 myeloma cells (ATCC, Rockville, Md.) and Raji cells.These cells are preferably cultured in Dulbecco's modified Eagle'sMedium (DMEM) supplemented with 10% FCS (Gibco/BRL, Gaithersburg,Mass.), 2 mM L-glutamine and 75 μg/ml gentamicin, (complete DMEM).Transfectomas are grown in Hybridoma Serum Free Medium, HSFM,(Gibco/BRL, Gaithersburg, Mass.) containing 10% of FCS and 75 μg/mlgentamicin (complete HSFM) or, where indicated, in HSFM containing onlyantibiotics. Selection of the transfectomas may be carried out incomplete HSFM containing 500 μg/ml of hygromycin (Calbiochem, San Diego,Calif.). All cell lines are preferably maintained at 37° C. in 5% CO₂.

[0043] An important aspect of this invention is that antibody variabledomains can be modeled by computer modeling (see, for example, Dion, inGoldenberg et al. eds., Cancer Therapy With Radiolabeled Antibodies, CRCPress, Boca Raton, Fla., 1994), which is incorporated by reference. Ingeneral, the 3-D structure for both the mLL22 and hLL2 mAbs are bestmodeled by homology. The high frequency of residue identities (75.0 to92.3%) between the deduced primary sequences of mLL2 light chain FRregions and human REI (VK) facilitates this approach because of theavailability of crystallographic data from the Protein Data Bank (PDRCode 1REI, Bernstein et al., J. Mol. Biol. 112: 535 (1977)), which isincorporated by reference. Similarly, antibody EU (VH) sequences can beselected as the computer counterparts for FR1 to FR3 of the mLL2 heavychain; FR4 was based on NEWM. As X-ray coordinate data is currentlylacking for the EU sequence, NEWM structural data (PDR Code 3FAB) forFRs 1 to 4 can be used, and amino acid side groups can be replaced tocorrespond to mLL2 or EU (hLL2) as needed. The CDR of the light chaincan be modeled from the corresponding sequence of 1MCP (L1 and L2) and1REI (L3). For heavy chain CDRs, H1 and H2 can be based on 2HFL and1MCP, respectively, while H3 can be modeled de novo. Wherever possible,side group replacements should be performed so as to maintain thetorsion angle between Cα and Cβ. Energy minimization may be accomplishedby the AMBER forcefield (Weiner et al, J. Amer. Chem. Soc. 106: 765(1984) using the convergent method. Potentially critical FR-CDRinteractions can be determined by initially modeling the light and heavyvariable chains of mLL2. All FR residues within a 4.5 Å radius of allatoms within each CDR can thereby be identified and retained in thefinal design model of hLL2.

[0044] Once the sequences for the hLL2 VK and VH domains are designed,CDR engrafting can be accomplished by gene synthesis using longsynthetic DNA oligonucleotides as templates and short oligonucleotidesas primers in a PCR reaction. In most cases, the DNA encoding the VK orVH domain will be approximately 350 bp long. By taking advantage ofcodon degeneracy, a unique restriction site may easily be introduced,without changing the encoded amino acids, at regions close to the middleof the V gene DNA sequence. For example, at DNA nucleotide positions157-162 (amino acid positions 53 and 54) for the hLL2 VH domain, aunique AvrII site can be introduced while maintaining the originallydesigned amino acid sequence (FIG. 4B). Two long non-overlappingsingle-stranded DNA oligonucleotides (˜150 bp) upstream and downstreamof the AvrII site (see, for example, oligo A and oligo B, Example 3below) can be generated by automated DNA oligonucleotide synthesizer(Cyclone Plus DNA Synthesizer, Milligen-Biosearch). As the yields offull length DNA oligonucleotides such as oligos A and B may be expectedto be low, they can be amplified by two pairs of flankingoligonucleotides (oligo Seq. ID Nos. 7 and 8 for oligo A; oligo Seq. IDNos. 9 and 10 for oligo B, Example 3) in a PCR reaction. The primers canbe designed with the necessary restriction sites to facilitatesubsequent subcloning. Primers for oligo A and for oligo B shouldcontain overlapping sequence at the AvrII site so that the resultant PCRproduct for oligo A and B, respectively, can be joined in-frame at theAvrII site to form a full length DNA sequence (ca 350 bp) encoding thehLL2 VH domain. The ligation of the PCR products for oligo A(restriction-digested with PstI and AvrII) and B (restriction-digestedwith AvrII and BstEII) at the AvrII site and their subcloning into thePstII/BstEII sites of the staging vector, VHpBS, can be completed in asingle three-fragment-ligation step (See, for example, Example 3). Thesubcloning of the correct sequence into VHpBS can be first analyzed byrestriction digestion analysis and subsequently confirmed by sequencingreaction according to Sanger et al., Proc. Natl. Acad. Sci. USA 74: 5463(1977).

[0045] The HindIII/BamHI fragment containing the Ig promoter, leadersequence and the hLL2 VH sequence can be excised from the staging vectorand subcloned to the corresponding sites in a pSVgpt-based vector, pG1g,which contains the genomic sequence of the human IgG constant region, anIg enhancer and a gpt selection marker, forming the final expressionvector, hLL2pG1g. Similar strategies can be employed for theconstruction of the hLL2 VK sequence. The restriction site chosen forthe ligation of the PCR products for the long oligonucloetides (oligos Cand D, see examples below) can be NruI in this case.

[0046] The DNA sequence containing the Ig promoter, leader sequence andthe hLL2 VK sequence can be excised from the staging vector VKpBR bytreatment with BamHI/HindIII, and can be subcloned into thecorresponding sites of a pSVhyg-based vector, pKh, which contains thegenomic sequence of human kappa chain constant regions, a hygromycinselection marker, an Ig and a kappa enhancer, forming the finalexpression vector, hLL2pKh.

[0047] As humanization sometimes results in a reduction or even loss ofantibody affinity, additional modification might be required in order torestore the original affinity (See, for example, Tempest et al.,Bio/Technology 9: 266 (1991); Verhoeyen et al., Science 239: 1534(1988)), which are incorporated by reference. Knowing that cLL2 exhibitsa binding affinity comparable to that of its murine counterpart (seeExample 5 below), defective designs, if any, in the original version ofhLL2 can be identified by mixing and matching the light and heavy chainsof cLL2 to those of the humanized version. SDS-PAGE analysis of thedifferent mix-and-match humanized chimeric LL2 under non-reducing (thedisulfide L-H chain connections remain intact) and reducing conditions(the chains separate, permitting analyses of the relationships of thedifferent types of light and heavy chains on the properties of themolecule). For example, migration as multiple bands as a higher apparentmolecular size can be due to the presence of a glycan group at theN-linked glycosylation site found at the FRI region of the murine VKdomain of LL2. For another example, a discrete band migrating at about25 kDa is the expected molecular size for a non-glycosylated lightchain.

[0048] In general, to prepare cLL2 mAb, VH and VK chains of mLL2 can beobtained by PCR cloning using DNA products and primers. Orlandi et al.,infra, and Leung et al., infra. The VK PCR primers may be subcloned intoa pBR327 based staging vector (VKpBR) as described above. The VH PCRproducts may be subcloned into a similar pBluescript-based stagingvector (VHpBS) as described above. The fragments containing the VK andVH sequences, along with the promoter and signal peptide sequences, canbe excised from the staging vectors using HindIII and BamHI restrictionendonucleases. The VK fragments (about 600 bp) can be subcloned into amammalian expression vector (for example, pKh) conventionally. pKh is apSVhyg-based expression vector containing the genomic sequence of thehuman kappa constant region. an Ig enhancer, a kappa enhancer and thehygromucin-resistant gene. Similarly, the about 800 bp VH fragments canbe subcloned into pG1g, a pSVgpt-based expression vector carrying thegenomic sequence of the human IgG1 constant region, an Ig enhancer andthe xanthine-guanine phosphoribosyl transferase (gpt) gene. The twoplasmids may be transfected into mammalian expression cells, such asSp2/0-Ag14 cells, by electroporation and selected for hygromycinresistance. Colonies surviving selection are expanded, and supernatantfluids monitored for production of cLL2 mAb by an ELISA method. Atransfection efficiency of about 1-10×10⁶ cells is desirable. Anantibody expression level of between 0.10 and 2.5 μg/ml can be expectedwith this system.

[0049] RNA isolation, cDNA synthesis, and amplification can be carriedout as follows. Total cell RNA can be prepared from a LL2 hybridoma cellline, using a total of about 10⁷ cells, according to Sambrook et al.,(Molecular Cloning: A Laboratory Manual, Second ed., Cold Spring HarborPress, 1989), which is incorporated by reference. First strand cDNA canbe reverse transcribed from total RNA conventionally, such as by usingthe SuperScript preamplification system (Gibco/BRL., Gaithersburg, Md.).Briefly, in a reaction volume of 20 μl, 50 ng of random primers can beannealed to 5 μg of RNAs in the presence of 2 μl of 10× synthesis buffer[200 mM Tris-HCl (pH 8.4), 500 mM KCl, 25 mM MgCl₂, 1 mg/ml BSA], 1 μlof 10 mM dNTP mix, 2 μl of 0.1 M DTT, and 200 units of SuperScriptreverse transcriptase. The elongation step is initially allowed toproceed at room temperature for 10 min followed by incubation at 42° C.for 50 min. The reaction can be terminated by heating the reactionmixture at 90° C. for 5 min.

[0050] The VK and VH sequences for cLL2 or hLL2 can amplified by PCR asdescribed by Orlandi et al., (Proc. Natl. Acad. Sci., USA, 86: 3833(1989)) which is incorporated by reference. VK sequences may beamplified using the primers CK3BH and VK5-3 (Leung et al.,BioTechniques, 15: 286 (1993), which is incorporated by reference),while VH sequences can be amplified using the primer CH1B which annealsto the CHI region of murine IgG, and VHIBACK (Orlandi et al., 1989above). The PCR reaction mixtures containing 10 μl of the first strandcDNA product, 9 μl of 10× PCR buffer [500 mM KCl, 100 mM Tris-HCl (pH8.3), 15 mM MgCl2, and 0.01% (w/v) gelatin] (Perkin Elmer Cetus,Norwalk, Conn.), can be subjected to 30 cycles of PCR. Each PCR cyclepreferably consists of denaturation at 94° C. for 1 min, annealing at50° C. for 1.5 min, and polymerization at 72° C. for 1.5 min. AmplifiedVK and VH fragments can be purified on 2% agarose (BioRad, Richmond,Calif.). See Example 3 for a method for the synthesis of an oligo A(149-mer) and an oligo B (140-mer) on an automated Cyclone Plus DNAsynthesizer (Milligan-Biosearch) for use in constructing humanized Vgenes.

[0051] PCR products for VK can be subcloned into a staging vector, suchas a pBR327-based staging vector VKpBR that contains an Ig promoter, asignal peptide sequence and convenient restriction sites to facilitatein-frame ligation of the VK PCR products. PCR products for VH can besubcloned into a similar staging vector, such as the pBluescript-basedVHpBS. Individual clones containing the respective PCR products may besequenced by, for example, the method of Sanger et al., Proc. Natl.Acad. Sci., USA, 74: 5463 (1977) which is incorporated by reference.

[0052] The DNA sequences described herein are to be taken as includingall alleles, mutants and variants thereof, whether occurring naturallyor induced.

[0053] The two plasmids can be co-transfected into an appropriate cell,e.g., myeloma Sp2/0-Ag14, colonies selected for hygromycin resistance,and supernatant fluids monitored for production of cLL2 or hLL2antibodies by, for example, an ELISA assay, as described below.

[0054] Transfection, and assay for antibody secreting clones by ELISA,can be carried out as follows. About 10 μg of hLL2pKh (light chainexpression vector) and 20 μg of hLL2pG1g (heavy chain expression vector)can be used for the transfection of 5×10⁶ SP2/0 myeloma cells byelectroporation (BioRad, Richmond, Calif.) according to Co et al., J.Immunol., 148: 1149 (1992) which is incorporated by reference. Followingtransfection, cells may be grown in 96-well microtiter plates incomplete HSFM medium (GIBCO, Gaithersburg, Md.) at 37° C., 5% CO₂. Theselection process can be initiated after two days by the addition ofhygromycin selection medium (Calbiochem, San Diego, Calif.) at a finalconcentration of 500 μg/ml of hygromycin. Colonies typically emerge 2-3weeks post-electroporation. The cultures can then be expanded forfurther analysis.

[0055] Transfectoma clones that are positive for the secretion ofchimeric or humanized heavy chain can be identified by ELISA assay.Briefly, supernatant samples (100 μl) from transfectoma cultures areadded in triplicate to ELISA microtiter plates precoated with goatanti-human (GAH)-IgG, F(ab′)₂ fragment-specific antibody (JacksonImmunoResearch, West Grove, Pa.). Plates are incubated for 1 h at roomtemperature. Unbound proteins are removed by washing three times withwash buffer (PBS containing 0.05% polysorbate 20). Horseradishperoxidase (HRP) conjugated GAH-IgG, Fc fragment-specific antibodies(Jackson ImmunoResearch, West Grove, Pa.) are added to the wells, (100μl of antibody stock diluted×10⁴, supplemented with the unconjugatedantibody to a final concentration of 1.0 μg/ml). Following an incubationof 1 h, the plates are washed, typically three times. A reactionsolution, [100 μl, containing 167 μg of orthophenylene-diamine (OPD)(Sigma, St. Louis, Mo.), 0.025% hydrogen peroxide in PBS], is added tothe wells. Color is allowed to develop in the dark for 30 minutes. Thereaction is stopped by the addition of 50 μl of 4 N HCl solution intoeach well before measuring absorbance at 490 nm in an automated ELISAreader (Bio-Tek instruments, Winooski, Vt.). Bound chimeric antibodiesare than determined relative to an irrelevant chimeric antibody standard(obtainable from Scotgen, Ltd., Edinburg, Scotland).

[0056] Antibodies can be isolated from cell culture media as follows.Transfectoma cultures are adapted to serum-free medium. For productionof chimeric antibody, cells are grown as a 500 ml culture in rollerbottles using HSFM. Cultures are centrifuged and the supernatantfiltered through a 0.2 micron membrane. The filtered medium is passedthrough a protein A column (1×3 cm) at a flow rate of 1 ml/min. Theresin is then washed with about 10 column volumes of PBS and proteinA-bound antibody is eluted from the column with 0.1 M glycine buffer (pH3.5) containing 10 mM EDTA. Fractions of 1.0 ml are collected in tubescontaining 10 μl of 3 M Tris (pH 8.6), and protein concentrationsdetermined from the absorbancies at 280/260 nm. Peak fractions arepooled, dialyzed against PBS, and the antibody concentrated, forexample, with the Centricon 30 (Amicon, Beverly, Mass.). The antibodyconcentration is determined by ELISA, as before, and its concentrationadjusted to about 1 mg/ml using PBS. Sodium azide, 0.01% (w/v), isconveniently added to the sample as preservative.

[0057] Comparative binding affinities of the mLL2, cLL2 and hcLL2antibodies thus isolated may be determined by direct radioimmunoassay.mLL2 can be labeled with ¹³¹I or ¹²⁵I using the chloramine T method(see, for example, Greenwood et al., Biochem. J., 89: 123 (1963) whichis incorporated by reference). The specific activity of the iodinatedantibody is typically adjusted to about 10 μCi/μg. Unlabeled and labeledantibodies are diluted to the appropriate concentrations using reactionmedium (HSFM supplemented with 1% horse serum and 100 μg/ml gentamicin).The appropriate concentrations of both labeled and unlabeled antibodiesare added together to the reaction tubes in a total volume of 100 μl. Aculture of Raji cells is sampled and the cell concentration determined.The culture is centrifuged and the collected cells washed once inreaction medium followed by resuspension in reaction medium to a finalconcentration of about 10⁷ cells/ml. All procedures are carried out inthe cold at 4° C. The cell suspension, 100 μl, is added to the reactiontubes. The reaction is carried out at 4° C. for 2 h with periodic gentleshaking of the reaction tubes to resuspend the cells. Following thereaction period, 5 ml of wash buffer (PBS containing 1% BSA) is added toeach tube. The suspension is centrifuged and the cell pellet washed asecond time with another 5 ml of wash buffer. Following centrifugation,the amount of remaining radioactivity remaining in the cell pellet isdetermined in a gamma counter (Minaxi, Packard Instruments, Sterling,Va.).

[0058] The Raji cell surface antigen binding affinities of mix-and-matchand fully humanized antibodies can be compared to that of cLL2 usingvarious concentrations of mLL2 F(ab′)₂ fragments devoid of the Fcportion as competitors, as evaluated by flow cytometry assay. Residualsurface-bound LL2 antibodies carrying the human Fe portions (cLL2 andmix-and-match LL2) can be detected by a FITC-labeled anti-human Fcspecific antibody in a flow cytometry assay. Where mix-and-match LL2antibodies exhibit antigen-binding affinities similar to that of cLL2,it can be concluded that the original designs for the humanization ofboth the light and heavy chains retain the mLL2 immunoreactivity.

[0059] The internalization of mLL2, cLL2 and hLL2 antibodies into targetcells can be followed by fluorescence labeling, essentially according tothe procedure of Pirker et al., J. Clin. Invest., 76: 1261 (1985), whichis incorporated by reference. Cultured Raji cells are centrifuged andthe cells resuspended in fresh medium to a concentration of about 5×10⁶cells/ml. To each well of a 96-well microtiter plate, 100 μl of the cellsuspension is added. The antibodies, 40 μg/ml, in a volume of 100 μl areadded to the reaction wells at timed intervals so as to terminate allreactions simultaneously. The plate is incubated at 37° C. in a CO₂ cellculture incubator. Unbound antibodies are removed by washing the cellsthree times with cold 1% FCS/PBS at the end of the incubation. The cellsare then treated with 1 ml of Formaid-Fresh [10% formalin solution(Fisher, Fair Lawn, N.J.)] for 15 min at 4° C. After washing, antibodiespresent either on the cell surface or inside the cells are detected bytreatment with FITC-labeled goat anti-mouse antibody (Tago, Burlingame,Calif.), or FITC-labeled goat anti-human antibody (JacksonImmunoResearch, West Grove, Pa.), depending on whether the antibodybeing assayed for is murine, chimeric, or humanized, respectively.Fluorescence distributions are evaluated using a BH-2 fluorescencemicroscope (Olympus, Lake Success, N.Y.).

[0060] The rate of antibody internalization can be determined accordingto Opresko et al., (J. Biol. Chem., 262: 4116 (1987)), usingradioiodinated antibody as tracer. Briefly, radiolabeled antibodies(1×10⁴ cpm) are incubated with the Raji cells (1×10⁶ cells/ml) at 4° C.for 2 h in 0.5 ml of DMEM medium containing 1% human serum. Followingthe reaction interval, non-specifically bound antibodies are removed bywashing three times with 0.5 ml of DMEM medium. To each of the reactiontubes 0.5 ml of DMEM medium is added and the suspension incubated at 37°C. for the determination of internalization. At timed intervals,triplicates of cells are removed and chilled immediately in an ice bathto stop further internalization. Cells are centrifuged at 1000×g for 5min at 4° C. The supernatant is removed and counted for radioactivity.The surface-bound radioactivity is removed by treatment with 1 ml 0.1 Macetate/0.1 M glycine buffer at pH 3.0 for 8 min. in the cold.Radioactivity removed by the acid treatment, and that remainingassociated with the cells, are determined. The ratio of theCPM_(internalization)/CPM_(surface) is plotted versus time to determinethe rate of internalization from the slope.

[0061] Detailed protocols for oligonucleotide-directed mutagenesis andrelated techniques for mutagenesis of cloned DNA are well-known. Forexample, see Sambrook et al. and Ausubel et al. above.

[0062] Asn-linked glycosylation sites may be introduced into antibodiesusing conventional site-directed oligonucleotide mutagenesis reactions.For example, to introduce an Asn in position 18 of a kappa protein, onemay alter codon 18 from AGG to AAC. To accomplish this, a singlestranded DNA template containing the antibody light chain sequence isprepared from a suitable strain of E. coli (e.g., dut⁻ung−) in order toobtain a DNA molecule containing a small number of uracils in place ofthymidine. Such a DNA template can be obtained by M13 cloning or by invitro transcription using a SP6 promoter. See, for example, Ausubel etal., eds., CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley & Sons,NY, 1987. An oligonucleotide containing the mutated sequence issynthesized conventionally, annealed to the single-stranded template andthe product treated with T4 DNA polymerase and T4 DNA ligase to producea double-stranded DNA molecule. Transformation of wild type E. coli(dut⁺ ung⁺) cells with the double-stranded DNA provides an efficientrecovery of mutated DNA.

[0063] Alternatively, an Asn-linked glycosylation site can be introducedinto an antibody light chain using an oligonucleotide containing thedesired mutation as the primer and DNA clones of the variable regionsfor the, VL chain, or by using RNA from cells that produce the antibodyof interest as a template. Also see, Huse, in ANTIBODY ENGINEERING: APRACTICAL GUIDE, Boerrebaeck, ed., W. H. Freeman & Co., pp 103-120,1992. Site-directed mutagenesis can be performed, for example, using theTRANSFORMER™ kit (Clontech, Palo Alto, Calif.) according to themanufacturer's instructions.

[0064] Alternatively, a glycosylation site can be introduced bysynthesizing an antibody chain with mutually priming oligonucleotides,one such containing the desired mutation. See, for example, Uhlmann,Gene 71: 29 (1988); Wosnick et al., Gene 60: 115 (1988); Ausubel et al.,above, which are incorporated by reference.

[0065] Although the general description above referred to theintroduction of an Asn glycosylation site in position 18 of the lightchain of an antibody, it will occur to the skilled artisan that it ispossible to introduce Asn-linked glycosylation sites elsewhere in thelight chain, or even in the heavy chain variable region.

[0066] The representative embodiments described below are simply used toillustrate the invention. Those skilled in these arts will recognizethat variations of the present materials fall within the broad genericscope of the claimed invention. The contents of all references mentionedherein are incorporated by reference.

EXAMPLE 1 Choice of Human Frameworks and Sequence Design for theHumanization of LL2 Monoclonal Antibody

[0067] By comparing the murine variable (V) region framework (FR)sequences of LL2 to that of human antibodies in the Kabat data base(Kabat et al., Sequences of Proteins of Immunological Interest, 5th ed.,U.S. Department of Health and Human Services, U.S. Government PrintingOffice, Washington, D.C.), which is incorporated by reference, the humanREI (FIG. 1A, Sequence ID No. 1) and EU (FIG. 1B, Sequence ID No. 2)sequences were found to exhibit the highest degree of sequence homologyto the FRs of VK and VH domains of LL2, respectively. Therefore, the REIand EU FRs were selected as the human frameworks onto which the CDRs forLL2 VK and VH were grafted, respectively. The FR4 sequence of NEWM,however, rather than that of EU, was used to replace the EU FR4 sequencefor the humanization of LL2 heavy chain. Based on the results ofcomputer modeling studies (FIGS. 2A and 2B), murine FR residues havingpotential CDR contacts, which might affect the affinity and specificityof the resultant antibody, were retained in the design of the humanizedFR sequences (FIG. 1).

[0068] Two versions of humanized heavy chain were constructed. In thefirst version (hLL2-1), the glutamine (Q) at amino acid position 5(Kabat numbering) was introduced to include a PstI restriction site tofacilitate its subcloning into the staging vector (FIG. 3). This murineresidue was converted, by oligo-directed mutagenesis, to the human EUresidue valine (V) in hLL2-2. It should be noted that in the originalmurine kappa chain variable sequence, a potential N-linked glycosylationsite was identified at positions 18-20 (FIG. 1) and was used forcarbohydrate addition. This glycosylation site was not included in theREI FR sequence used for LL2 light chain humanization.

[0069] See Example 3 for more oligonucleotide detail.

EXAMPLE 2 PCR Cloning and Sequence Elucidation for LL2 Heavy and LightChain Variable Regions

[0070] The variable regions for both heavy (VH) and light (VK) chains ofmLL2 (IgG2a) were obtained by PCR cloning using DNA primers as describedin general above and in greater detail in Example 3, below. As PCR isprone to mutations, the variable region sequence of multiple individualclones for either the heavy or light chains was determined for sixclones and confirmed to be identical prior to use for the constructionof the chimeric antibody.

[0071] The PCR products for VK were subcloned into a pBR327-basedstaging vector, VKpBR, which contained an Ig promoter, a signal peptidesequence and convenient restriction sites to facilitate in-frameligation of the VK PCR products (FIG. 3A). The PCR products for VH weresubcloned into a similar pBluescript-based staging vector, VHpBS (FIG.3B).

[0072] As noted above, at least six individual clones containing therespective PCR products were sequenced according to the method of Sangeret al., 1977, above. All were shown to bear identical sequences andtheir respective sequences were elucidated, as shown in FIG. 4A for LL2VK (Sequence ID NO. 3) and in FIG. 4B for LL2 VH (Sequence ID NO. 4). Nodefective mutations were identified within the sequences encoding the VKand VH regions. Comparison of the PCR-amplified variable regionsequences of LL2 with the Kabat database (Kabat et al., above) suggestedthat the VK and VH sequences of LL2 belong to subgroup 5 and 2B,respectively. Important residues such as Cys for intra-domain disulfidelinkage were retained at appropriate positions.

[0073] In the FRI framework region of VK, an N-linked carbohydrateattachment site, Asn-Val-Ser, was identified at position 18-20 (FIG.4A), suggesting that the VK of LL2 might be glycosylated. As will bedetailed below, SDS-PAGE analysis under reducing conditions demonstratedthat this Asn glycosylation site is indeed utilized for carbohydrateaddition. The presence of the glycosylation site in the variable regiondoes not, however, appear to affect the immunoreactivity of theantibody. A comparison of the immunoreactivity of mLL2 with that of cLL2in a competitive RIA showed that the two antibodies have nearlyidentical activities.

EXAMPLE 3 PCR/Gene Synthesis of the Humanized V Genes

[0074] The designed sequence for the hLL2 VH domain, the construction ofthe hLL2 VH domain by long oligonucleotides and PCR, and the stagingvector VHpBS containing the hLL2 VH domain are summarized in the sketchshown in FIG. 6.

[0075] For the construction of the hLL2 VH domain, oligo A (149-mer) andoligo B (140-mer) were synthesized on an automated CYCLONE PLUS™ DNAsynthesizer (Milligen Bioresearch).

[0076] Oligo A (Sequence ID No. 7 below) represents the minus strand ofthe hLL2 VH domain complementary to nt 24 to 172.

[0077] Sequence ID No. 7 5′-TAT AAT CAT TCC TAG GAT TAA TGT ATC CAA TCCATT CCA GAC CCT GTC CAG GTG CCT GCC TGA CCC AGT GCA GCC AGT AGC TAG TAAAGG TGT AGC CAG AAG CCT TGC AGG AGA CCT TCA CTG ATG ACC CAG GTT TCT TGACTT CAG CC-3′

[0078] Oligo B (Sequence ID No. 8 below) represents the minus strand ofthe hLL2 VH domain complementary to nt 180 to 320.

[0079] Sequence ID No. 8 5′-CCC CAG TAG AAC GTA ATA TCC CTT GCA CAA AAATAA AAT GCC GTG TCC TCA GAC CTC AGG CTG CTC AGC TCC ATG TAG GCT GTA TTGGTG GAT TCG TCT GCA GTT ATT GTG GCC TTG TCC TTG AAG TTC TGA TT-3′

[0080] Oligos A and B were cleaved from the support and deprotected bytreatment with concentrated ammonium hydroxide. After the samples werevacuum-dried (SpeedVac, Savant, Farmingdale, N.Y.) and resuspended in100 μl of water, incomplete oligomers (less than 100-mer) were removedby centrifugation through a CHROMOSPIN-100™ column (Clonetech, PaloAlto, Calif.) before the DNA oligomers were amplified by PCR. Allflanking primers for the separate amplifications and PCR cloning ofoligos A and B were purified by SDS-PAGE essentially according to themethods of Sambrook et al., 1989, above. From the CHROMASPIN-purifiedoligo A, 1 μl of sample stock was PCR-amplified in a reaction volume of100 μl by adding 5 μl of 5 μM of oligo Sequence ID No. 9:

[0081] 5′-CCA GCT GCA GCA ATC AGG GGC TGA AGT CAA GAA ACC TG-3′

[0082] and oligo Sequence ID No. 10:

[0083] 5′-AAG TGG ATC CTA TAA TCA TTC CTA GGA TTA ATG-3′

[0084] in the presence of 10 μl of 10×PCR Buffer (500 mM KCl, 100 mMTris.HCL buffer, pH 8.3, 15 mM MgCl₂) and 5 units of AMPLITAQ™ DNApolymerase (Perkin Elmer Cetus, Norwalk, Conn.). This reaction mixturewas subjected to 30 cycles of PCR reaction consisting of denaturation at94° C. for 1 minute, annealing at 50° C. for 1.5 minutes, andpolymerization at 72° C. for 1.5 minutes.

[0085] Oligo B was PCR-amplified by the primer pairs Sequence ID No. 11:

[0086] 5′-TAA TCC TAG GAA TGA TTA TAC TGA GTA CAA TCA GAA CTT CAA GGACCA G-3′

[0087] and Sequence ID No. 12:

[0088] 5′-GGA GAC GGT GAC CGT GGT GCC TTG GCC CCA GTA GAA CGT AGT AA-3′

[0089] under similar conditions.

[0090] Double-stranded PCR-amplified products for oligos A and B weregel-purified, restriction-digested with PstI/AvrII (PCR product of oligoA) and BstEII/AvrII (PCR product of oligo B), and sucloned into thecomplementary PstI/BstEII sites of the heavy chain staging vector,VHpBS. The humanized VH sequence was subcloned into the pG1g vector,resulting in the final human IgG1 heavy chain expression vector,hLL2pG1g.

[0091] For constructing the full length DNA of the humanized VKsequence, oligo E (150-mer) and oligo F (121-mer) were synthesized asdescribed above.

[0092] Oligo E Sequence ID No. 13: 5′-CCT AGT GGA TGC CCA GTA GAT CAGCAG TTT AGG TGC TTT CCC TGG TTT CTG GTG GTA CCA GGC CAA GTA GTT CTT GTGATT TGC ACT GTA TAA AAC ACT TTG ACT GGA CTT ACA GCT CAT AGT GAC CCT ATCTCC AAC AGA TGC GCT CAG-3′

[0093] represents the minus strand of the humanized VK domaincomplementary to nt 31 to 180, and this sequence was PCR-amplified byoligo Sequence ID No. 14:

[0094] 5′-GAC AAG CTT CAG CTG ACC CAG TCT CCA TCA TCT CTG AGC GCA TCTGTT GGA G-3′

[0095] and oligo Sequence ID No. 15

[0096] 5′-AGA GAA TCG CGA AGG GAC ACC AGA TTC CCT AGT GGA TGC CCAGTA-3′.

[0097] Oligo F Sequence ID No. 16: 5′-GCA CCT TGG TCC CTC CAC CGA ACGTCC ACG AGG AGA GGT ATT GGT GAC AAT AAT ATG TTG CAA TGT CTT CTG GTT GAAGAG AGC TGA TGG TGA AAG TAA AAT CTG TCC CAG ATC CGC TGC C-3′

[0098] represents the minus strand of the humanized LL2 VK domaincomplementary to nt 208 to 327, and was PCR amplified by oligo SequenceID No. 17:

[0099] 5′-GAC AAG CTT TCG CGA TTC TCT GGC AGC GGA TCT GGG ACA G-3′

[0100] and oligo Sequence ID No. 18:

[0101] 5′-GAC CGG CAG ATC TGC ACC TTG GTC CCT CCA CCG-3′.

[0102] Gel-purified PCR products for oligos E and F wererestriction-digested with PvuII/NruI and NruI/BglII, respectively. Thetwo PCR fragments E and F were then joined at the NruI site and ligatedto the complementary PvuI/BcII sites of the light chain staging vector,VKpBR. The humanized VK sequence was subcloned into vector pKh to formthe final human kappa chain expression vector, hLL2pKh.

[0103] To express the humanized antibodies, about 10 μg of linearizedhLL2pKh and 20 μg of linearized hLL2pG1g were used to transfect 5×10⁶SP2/0 cells by electroporation. The transfectomas were selected withhygromycin at 500 μg/ml and secreted antibody was purified on a 1×3 cmcolumn of protein A. After concentrating the purified antibody byCentricon 30 centifugation, antibody concentration was determined byELISA. The final concentration of the antibody was adjusted to 1 mg/mlin PBS buffer containing 0.01% (w/v) sodium azide as a preservative.

[0104] In FIG. 1 (Sequence ID Nos. 1 and 2), there is compared the aminoacid sequence between murine and humanized LL2 VK domains (FIG. 1A) andbetween murine and humanized LL2 VH domains (FIG. 1B). In the VK chain,human REI framework sequences were used for all FRs. In the VH chain,human EU framework sequences were used for FR 1-3, and NEWM sequenceswere used for FR-4. Only human FR sequences that are different from thatof the mouse are shown. Asterisks indicate murine FR sequences that aredifferent from that of the human FR at corresponding positions. Murineresidues at these positions were retained in the humanized structure.CDRs are boxed.

[0105] In FIG. 4A (Sequence ID No. 3) there are shown the doublestranded DNA and corresponding amino acid sequences (shown by singleletter code) of the humanized LL2 VK domain. CDR 1-3 amino acidsequences are boxed. The corresponding display for VH is shown in FIG.4B (Sequence ID No. 4).

[0106] In FIG. 5A (Sequence ID No. 5) and FIG. 5B (Sequence ID No. 6)there are shown double-stranded DNA sequences and amino acid sequencesof humanized LL2 VK and LL2 VH, respectively. Amino acid sequences areshown by the single-letter code, and CDR amino acid sequences are boxed.

EXAMPLE 4 Construction, Expression and Purification of Chimeric LL2Antibodies

[0107] The fragments containing the VK and VH sequences of LL2, togetherwith the promoter and signal peptide sequences, were excised fromLL2VKpBR and LL2VHpBS, respectively, by double restriction digestionwith HindIII and BamHI. The about 600 bp VK fragments were thensubcloned into the HindIII/BamHI site of a mammalian expression vector,pKh (FIG. 3A). pKh is a pSVhyg-based expression vector containing thegenomic sequence of the human kappa constant region, an Ig enhancer, akappa enhancer and the hygromycin-resistant gene. Similarly, the ca. 800bp VH fragments were subcloned into the corresponding HindIII/BamHI siteof pG1g (FIG. 3B), a pSVgpt-based expression vector carrying the genomicsequence of the human IgG1 constant region, an Ig enhancer and thexanthine-guanine phosphoribosyltransferase (gpt) gene. The finalexpression vectors are designated as LL2pKh and LL2pG1g, respectively.

[0108] The two plasmids were co-transfected into Sp2/0-Ag14 cells byelectroporation and selected for hygromycin resistance. Supernatantsfrom colonies surviving selection were monitored for chimeric antibodysecretion by ELISA assay (see above). The transfection efficiency wasapproximately 1-10×10⁶ cells. The antibody expression level, in aterminal culture, was found to vary in the range between <0.10 and 2.5μg/ml.

[0109]FIG. 7 shows the results of analyzing protein A-purified mLL2(lanes 4 and 7) and cLL2 (lanes 5 and 8) by SDS-PAGE under reducing andnonreducing conditions, respectively. HMW stands for high molecularweight protein markers, and LMW for light molecular weight markers. Thelight chains of both mLL2 and cLL2 (lanes 4 and 5) migrated primarily asa doublet band, with a higher than expected apparent molecular weight.As the human kappa constant region of cLL2 is known to contain nopotential glycosylation site, it can be inferred that the potentialglycosylation site identified in the FR1 region of LL2 VK domain wasutilized.

[0110]FIG. 8 shows the results of analyzing different versions of hLL2and cLL2 antibodies by SDS-PAGE under reducing and non-reducingconditions. As before, LMW and HMW are molecular weight markers. Lanes 3and 6 are cLL2 antibodies. Lanes 4 and 7 are hLL2 with seven murine FRresidues in the VH domain (hLL2-1). Lanes 5 and 8 are hLL2 with 6 murineFR residues in the VH domain (hLL2-2). The humanized light chainsmigrated more rapidly and as more discrete bands compared to chimericlight chains.

[0111]FIG. 9 shows the results of SDS-PAGE analysis on mix-and-match andcLL2 and hLL2 antibodies under both reducing and non-reducingconditions. Lanes 1 and 2 are molecular weight markers. Lanes 3 and 7are cLL2. Lanes 4 and 8 are mix-and-match with a humanized light andchimeric heavy chain [(hL/cH)LL2]. Lanes 5 and 9 are chimeric light andhumanized heavy (Version 1) chains [(cL/hH)LL2-1]. Lanes 6 and 10 arechimeric light and a humanized heavy (version 2) chains [(cL/hH)LL2-2].The humanized LL2 version 1 contains 7 murine FR residues in the VHdomain, while version 2 contains 6 murine FR residues in the VH domain.It is noteworthy that the position of the light chain of (hL/cH)LL2(lane 4) is different from that of the others, suggesting that there isno carbohydrate attachment to the humanized LL2 light chain.

EXAMPLE 5 Binding of cLL2 Antibody to Raji Cell Surface Antigens

[0112] A competition cell binding assay was carried out to assess theimmunoreactivity of cLL2 relative to the parent mLL2. Using ¹³¹I-labeledmLL2 (0.025 μg/ml) as a probe, Raji cells were incubated with theantibodies and the relative binding to the cells determined from theamount of cell-bound labeled mLL2 (see above). As shown by thecompetition assays described in FIG. 10, both mLL2 and cLL2 antibodiesexhibited similar binding activities.

[0113] The results were confirmed by a second competition assay based onflow cytometry. Briefly, using Raji cells as before and varying theconcentration of one antibody relative to other, as before, the amountof bound mLL2 or cLL2 was determined with FITC-labeled anti-mouse Fc oranti-human Fc antibodies followed by analysis using flow cytometry.

EXAMPLE 6 Binding of hLL2 Antibodies to Raji Cells

[0114] In experiments similar to those of Example 5, the antigen bindingaffinities of the three different combinations of mix-and-match orhumanized LL2 were compared with that of cLL2 in the flow cytometryassay.

[0115] Briefly, 1 μg of cLL2, mix-and-match LL2, hLL2-1 or hLL2-2antibodies were incubated with 10⁸ Raji cells in the presence of varyingconcentrations of mLL2 F(ab′)₂ fragments (as competitor) in a finalvolume of 100 μl of PBS buffer supplemented with 1% FCS and 0.01% sodiumazide. The mixture was incubated for 30 minutes at 4° C., and washedthree times with PBS to remove unbound antibodies. By taking advantageof the presence of human Fc portions in the antibodies, the bindinglevels of the antibodies were assessed by adding a 20× dilutedFITC-labeled goat anti-human IgG1, Fc fragment-specific antibodies(Jackson ImmunoResearch, West Grove, Pa.). The cells were washed threetimes with PBS, and fluorescence intensities measured by a FACSCANfluorescence activated cell sorter (Becton-Dickinson, Bedford, Mass.).The results are shown in FIG. 11A.

[0116] Using the same methods, cLL2 was compared to two versions of hLL2(FIG. 11B).

[0117] The results shown in FIGS. 11A and B demonstrate that theimmunoreactivity of cLL2 is similar or identical to that of humanized ormix-and-match antibodies. Taken together with the comparison of cLL2with mLL2 (FIG. 10), the authenticity of the sequences for chimeric andhumanized VK and VH obtained is established, and the functionality ofcLL2 and hLL2 confirmed.

EXAMPLE 7 Internalization of mLL2 and cLL2 by Raji Cells

[0118] One of the unique characteristics of the LL2 antibody is itsrapid internalization upon binding to Raji cells (Shih et al., 1994above). Murine LL2 after internalization is likely to be rapidlytransferred to the Golgi apparatus and from there to the lysosomes, theorganelle responsible for the degradation of a wide variety ofbiochemicals (Keisari et al., Immunochem., 10: 565 (1973)).

[0119] Rates of antibody internalization were determined according toOpresko et al., 1987 above. The ratio ofCPM_(intracellular)/CPM_(surface) was determined as a function of time.

[0120] Rates of LL2 antibody internalization were determined byincubating radiolabeled LL2 antibody (1×10⁶ cpm) with 0.5×10⁶ Raji cellsin 0.5 ml of DMEM buffer containing 1% human serum for 2 hrs. at 4° C.Excess human serum was included to saturate Raji cell surface Fcreceptors in order to exclude or minimize non-antigen-specificinternalization mediated through the Fc receptors. Unbound radiolabeledLL2 antibodies were removed from the cells by washing three times with0.5 ml portions of DMEM at 4° C. Cells were then incubated at 37° C.,and, at timed intervals, aliquots of the cell suspension weretransferred to ice in order to stop internalization. The cells in thesealiquots were isolated by centrifugation at 1,000×g for 5 mins. at 4°C., and surface bound radiolabeled LL2 stripped off cells with 1 ml of0.1 M glycine acetate buffer, pH 3, for 8 mins. at 4° C. Radioactivitythus obtained (CPM surface) and radioactivity remaining in the cells(CPM intracellular) were determined. Rates of internalization werecalculated from the slope of the plot of intracellular:surfaceradioactivity ratios as a function of time.

[0121] As shown in FIG. 12, mLL2, cLL2, cLL2Q and hLL2 antibodies wereinternalized at a similar rate (Ke=0.107 (mLL2) to 0.1221 (cLL2Q, NVT toQVT mutation). Those numbers suggested that approximately 50% of thesurface-bound antibody could be internalized in 10 min. The results showthat neither chimerization nor humanization nor deglycosylation bymutagenesis of mLL2 antibodies impair rates of internalization.

[0122] The pattern of internalization for mLL2, cLL2 and hLL2 was alsomonitored by fluorescence microscopy on a time-course basis using aFITC-labeled second antibody probe as described in the specification.Internalization of both antibodies was observed in at the earliest timepoint measurable. At 5 minutes, antibodies were seen both on the cellsurface and internalized in areas immediately adjacent to the membraneas cytoplasmic micro-vesicles. At 15 min. post-incubation, the fine dotsdispersed around the intramembrane began to merge into a group ofgranules, at locations believed to be the Golgi apparatus. As moreantibodies were being internalized after 30 min. of incubation,redistribution of the grouped antibodies to scattered locations,probably the lysosomes in which the antibodies were degraded, wasobserved. At 2 hrs post-incubation, most of the antibodies were foundinside the cell. Only strong surface staining was observed when LL2 wasincubated for 20 min on ice. Both mLL2 and cLL2 were internalized with asimilar pattern. The internalization of LL2 was associated specificallywith antigen-antibody binding, as the irrelevant control humanizedantibody demonstrated only dull surface staining.

[0123] A103 antibody (an IgG2a antibody that binds to the surface of allhuman epithelial cells but does not internalize efficiently (Mattes etal., Hybridoma, 2: 253 (1983)) showed strong membrane staining at up to2 h, while the anti-transferrin receptor antibody (5F9) internalizedrapidly, just as did LL2.

EXAMPLE 8 Role of Glycosylation Site in FRI Region of LL2 VK Sequence

[0124] Of particular inventive interest is the identification of anAsn-glycosylation site at position 18-20 within the FRI region of theLL2 NVT light chain sequence (FIG. 4A). As shown above, SDS-PAGEanalysis under reducing condition suggests that the Asn glycosylationsite is utilized for carbohydrate addition.

[0125] In this example, the influence of the carbohydrate moiety atposition 18-20 on the functional activities of the light chains wasexamined.

[0126] Murine and chimeric LL2 light chains were treated with (+) orwithout (−) endoglycosidase F conventionally, and the antibody productsexamined by SDS-PAGE under reducing and non-reducing conditions (FIG.13). There was no distinction between the antibody types as toelectrophoretic behavior. In both cases, deglycosylation reduced therate of migration of the light chain.

[0127] The effect of deglycosylation on the binding affinity to Rajicells of the mLL2 antibody is shown in FIG. 14. Removing carbohydrate byendoglycosidase F was without influence on the binding activity.

[0128] A mutation was introduced at position 18 of the light chain sothat the Asn was replaced with Gin to produce LL2Q VK FR1. SDS-PAGEanalyses demonstrated that the NVT to QVT mutation abolishedglycosylation of the antibody. Comparison of the Raji cell bindingaffinity for cLL2 with and without light chain VK glycosylationdemonstrated that the carbohydrate moiety was without influence onbinding of the antibody to these cells.

[0129] It can be concluded that the presence of the carbohydrate site inthe variable region does not affect the immunoreactivity of theantibody. Computer modeling studies suggested that the VK carbohydratemoiety in LL2 is remotely positioned from the CDRs and forms a “cap”over the bottom loops of the FR-associated B-barrels supporting theCDRs.

[0130] Humanization without inclusion of the original glycosylation siteresulted in a CDR-grafted LL2 antibody with immunoreactivity comparableto that of its murine counterpart.

[0131] These characteristics indicate that the glycosylation site can beused for conjugating therapeutic or diagnostic agents to LL2 withoutcompromising the ability of the antibody to bind and internalize inB-lymphoma or leukemia cells.

EXAMPLE 9 Conjugation of LL2 at its Carbohydrate-Bearing Site

[0132] The apparent lack of involvement of the variable regioncarbohydrate moiety in the functional activities of mLL2, cLL2 and hLL2mAbs indicates that this moiety could profitably be used as the site ofattachment of cytotoxic or detection agents such as radionuclides ortoxins, and thereby avoid potential interference with the binding of theconjugate to a cell surface.

[0133] Using procedures described in Shih et al., U.S. Pat. No.5,057,313 (which is incorporated by reference) for preparing antibodyconjugates through an oxidized carbohydrate moiety of the antibody and aprimary alkylamino group of a polymeric carrier to which are covalentlyone or more of a variety of drugs, toxins, chelators and detectablelabels, a doxorubicin-dextran-LL2 antibody fragment devoid of appendedglycans was produced containing multiple copies of the drug. Thecarbohydrate moieties of the cLL2 VK FR1 region involved were thosecovalently bound to the Asn glycosylation site.

[0134] In one synthesis, dextran (18-40 kDa) was converted to an aminodextran by oxidation of the dextran by NaIO₄, Schiff base formation withNH₂—CH₂—CHOH—CH₂—NH₂, and reduction with NaBH₄. The amino dextran wasthen condensed with doxorubicin (DOX) in the presence of succinicanhydride and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide to produceDOX-aminodextran. The latter was then condensed with an aldehydic groupon LL2 VK FR-1 produced by oxidizing the carbohydrate moiety of theantibody fragment with NaIO₄.

[0135] In one preparation of DOX-LL2, the number of moles of DOXattached to dextran was 14 moles per mole dextran, and the number ofmoles of doxorubicin per mole F(ab′)2 was 8.9. The immunoreactivity inthe Raji cell binding assay above was about 80% of control values.

[0136] This conjugation system is not limited to the mLL2 antibody. In acomparative study, 15-19 moles of DOX/mole of cLL2 were bound.

[0137] The conjugation possibilities are not limited to the use of acarrier dextran as in the example above. For example, the carbohydratemoiety of the LL2 VK FR1 region can be oxidized to produce aldehydicgroups. These in turn can be reacted with an amino group on any drug toproduce a Schiff base which, upon reduction, produces multiple copies ofthe drug stably linked to the antibody via alkyamine groups.

[0138] For example, where the drug is aminohexyl DTPA (a chelatingagent), there is produced a LL2 covalently bound to a chelator. Thechelator can be used to deliver to target tissues, for example, aradionuclide or paramagnetic metal ion, with a potential for diagnosticand therapeutic uses. DTPA-LL2 conjugates were produced containing 5.5moles of the chelator/mole of antibody which, in turn, chelated 47.3% ofY-90 and 97.4% In-111

[0139] It should be emphasized that the above-described examples merelydescribe several specific embodiments of the invention, and applicantsdo not intend to be limited as to scope of claims by these specificexamples.

[0140] Applicants also incorporate by reference all publications andpatents cited in the specification.

We claim:
 1. An immunoconjugate comprising an antibody or a fragmentthereof specific for B-cells which is conjugated to Y-90 through a DTPAchelating agent.
 2. The immunoconjugate of claim 1, wherein the DTPAchelating agent is aminohexyl DTPA.
 3. The immunoconjugate of claim 1,wherein at least 47.3% of Y-90 is chelated.
 4. The immunoconjugate ofclaim 1, wherein the immunoconjugate is internalizing.
 5. Theimmunoconjugate of claim 4, wherein conjugation through the DPTAchelating agent to Y-90 does not reduce binding to B-cells orinternalization function relative to the unconjugated antibody.
 6. Amethod of treating B-cell lymphoma and lymphocytic leukemia comprisingadministering an immunoconjugate as claimed in claim 1 to a subjectsuffering from B-cell lymphoma or lymphocytic leukemia.
 7. A method oftreating B-cell lymphoma and lymphocytic leukemia comprisingadministering an immunoconjugate as claimed in claim 2 to a subjectsuffering from B-cell lymphoma or lymphocytic leukemia.
 8. A method oftreating B-cell lymphoma and lymphocytic leukemia comprisingadministering an immunoconjugate as claimed in claim 3 to a subjectsuffering from B-cell lymphoma or lymphocytic leukemia.
 9. A method oftreating B-cell lymphoma and lymphocytic leukemia comprisingadministering an immunoconjugate as claimed in claim 4 to a subjectsuffering from B-cell lymphoma or lymphocytic leukemia.
 10. A method oftreating B-cell lymphoma and lymphocytic leukemia comprisingadministering an immunoconjugate as claimed in claim 5 to a subjectsuffering from B-cell lymphoma or lymphocytic leukemia.